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Fig. 3. Inserts with cohesive ends complementary to the ends of the
linearized Clone-it™ vectors generated by staggered PCR.
2)
Clone-it™ shRNA Cloning/Expression Vectors:
Two approaches have been developed for in vivo expression of siRNA from plasmid and viral vectors. In one approach, the
sense and antisense strands are transcribed separately from two independent promoters and form the siRNA duplex (Lee 2002,
Miyagishi 2002). The second approach, as employed with the Clone-it™ shRNA Cloning/Expression Vectors, uses a singlestranded shRNA sequence with a fold-back stem-loop structure (also known as a “hairpin”) that is expressed from a single
promoter (Abbas-Terki 2002, Qin 2003, Wiznerowicz 2003). This sequence is then converted into double-stranded siRNA after
intracellular processing cleaves the loop (Brummelkamp 2002, Paddison 2002). In both approaches, the siRNA molecules are
transcribed from constitutive RNA polymerase III promoters (i.e., U6 and/or H1) and terminated with TTTTT (T5) sequences
(Tuschl 2002). The U6 and H1 promoters are different in size but contain the same conserved sequence elements (Myslinski
2001).
In the Clone-it™ shRNA Cloning and Expression Vectors, the RNA polymerase III H1 promoter is used to express the shRNA
sequence. The shRNA template oligonucleotides are cloned into the Ligase-free Cloning Site (LCS) located just downstream of
the H1 promoter (Figure 1). The linearized vectors provided contain two unique 5’ overhangs to facilitate directional cloning of
shRNA template oligos (Figure 4). When the shRNA construct is expressed from the constitutive H1 promoter and terminated
with the TTTTT sequence, the shRNA transcript folds into the hairpin structure, which is recognized by the DICER enzyme,
cleaved to form a functional double-stranded siRNA and transferred to a RISC complex for selective digestion of complementary
target mRNAs (Figure 4).
Fig. 4. Example shRNA construct targeting the p53 gene. The nucleotides
for the specific siRNA sequence targeting the p53 gene are shown in capital
letters. The shRNA sense and antisense sequences flank the region coding for
the loop structure. In addition, the terminator sequence ttttt for the RNA
polymerase III is included after the antisense portion. The Forward and Reverse
arrows refer to the PCR primers contained in the vector to confirm positive
clones. After transcription, a stem-loop-stem shRNA molecule is produced. This
molecule is processed by the DICER enzyme to generate a double-stranded
siRNA effector.
In order to clone such an shRNA expression cassette into the Clone-it™ shRNA expression vectors, instead of synthesizing two
65-nt oligos required for the conventional ligase-dependent method, we recommend the design and synthesis of four short oligos
as depicted in Fig. 5. This approach is more cost-effective and is more accurate and efficient. The oligos are then mixed, heat
and cooled to form the nicked, cohesive ended shRNA insert (Fig. 5).
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